Pyrogen removal from streptomycin salts



rmasoeeaiaso UNITED sTATes PATEN omen-,1 assess: y

Eugene E. Howe, Linden, and Tishler, Bah- No Drawing. Application November 8, 1948,

Serial No. 708,818

Claims. (Cl. 260-210) 1 This invention relates to the removal of pyrog'ens from therapeutic preparations and particularly to a process for the removal-of pyrogens from solutions of streptomycin to obtain solutions suitable for parenteral use. 5 about 25% of an acid salt of streptomycin can The physiological reactions caused by the presbe employed although it is preferable to use more ence of pyrogens in therapeutic preparations are dilute solutions, 1. e., solutions containing 2 to particularly objectionable in the treatment of of streptomycin acid salt. These solutions severe infection or disease because the patient recan be either solutions obtained in usual puriceiving treatment is often so weakened by the inficatio procedures or solutions prepared by disfcction that these reactions will prevent or greatsolving solid streptomycin acid salt in water, ly hinder recovery. Streptomycin has been dempreferably pyrogen-free. onstrated to be a very powerful drug for com- For removing pyrogens from such solutions an hating stubborn infections and diseases, but one amount of powdered charcoal such as "Darco of the factors hindering its more extended use 6-60" or the like, equal to about 12 to 50% by has been the diillculty in keeping the pyrogen weight, based upon the weight of streptomycin content of streptomycin preparations sufficiently acid salt. is added to the solution and the mixture low to prevent objectionable reactions. is agitated for about one hour. The mixture is Attempts havebeen made to remove pyrogens then filtered to remove the charcoal, preferably from streptomycin by contacting with glass wool, using a filter cloth to facilitate recovery of the filtering through ertel discs (asbestos fibers), or charcoal for re-slurring. After. filtration, the by prolonged contact with shredded ertel discs. charcoal is returned to the original container. These procedures, however, have little or no efslurried with pyrogen-free water equal in volfect in reducing the pyrogen content of streptoume to about 10% of the volume of the original mycin preparations. solution treated. This mixture is then filtered as It is now discovered, in accordance with the before and the filtrate is combined with the origpresent invention, that pyrogens can be effectiveinal filtrate. (The washing of the charcoal can ly removed from acid salts of streptomycin, such also be done in the filter instead of by removal as streptomycin hydrochloride, streptomycin suland re-slurrying, if desired.) The charcoal can fate, and the like, in aqueous solution by treatbe again washed or re-slurried, if desired, to furment with powdered charcoal such as Darco ther reduce the amount of Streptomycin remain- G-BO or other commercially available charcoals. ing on the charcoal, and after the final filtra- At the same time, some improvement in purity tion, the charcoal is set aside for recovery of the is obtained in this process. It is surprising and residual adsorbed streptomycin. (All the ceramunexpected that powdered charcoal could be used n glassware d in h fll ra lons. and in for this purpose in view of the fact that strept'ocollecting and handling the filtrates should be mycin itself is so readily adsorbed upon charcoal made pyrogen-free by heating to 250 C. for one as, for example, in the original recovery of strephour.) The combined filtrates are then sterilized tomycin from culture broth containing the same by filtration through a Seitz filter, and then (SCImtZ. Bugle. and waksman, Proceedings of the 40 dried. pre b y by f e g and dryin om the Society for lhrperimental Biology and Medicine, frozen state under vacuum. 55, 66-69 (1944) It is found, however, that The amount of charcoal to be used varies with upon treatment of an aqueous solution of an acid the concentration of the streptomycin sol i salt of streptomycin with charcoal, very little 10 to 12% of charcoal generally being sufficient of the streptomycin is adsorbed on the charcoal. with a 2% solution of streptomycin acid salt, and This is apparently due to the slight acidity of about 25 to of charcoal being required with the acid salts such as streptomycin hydrochloride, a 25% solution of streptomycin acid salt. It is streptomycin sulfate, and the like in aqueous soto be understood, however, that diflerent lots of lution. It should be noted that the small amount streptomycin vary considerably in pyrogen conof streptomycin which is adsorbedis not lost, but 50 t nd th in some sta ces e Opt mum can be recovered from the charcoal by elution amount of charcoal may fall outside the ranges with alcoholic acid, or better, the streptomycin above mentioned. can be almost completely removed from the char- The time of contact of the streptomycin soluooal by slurrying with water. The aqueous streption with charcoal is relatively critical. If the tomcin solution thus recovered can be processed time 91 contact 15 appreciably less than an hour,

. 2 by itself or can be used to dissolve another batch for pyrogen treatment.

Incarrying out the process of the present invention, aqueous solutions containing as much as contain about 10 to 12%.

assuming the proper amount of charcoal is used, the pyrogens are not completely adsorbed, and prolonging the time of contact appreciably beyond one hour has not been found to be of any advantage.

The removal of pyrogens appears to proceed well with untreated charcoal, preheated charcoal, or acid-washed charcoal, although there is believed to be a slight advantage in using charcoal which has been preheated to 250 C. for about eight hours.

The following examples show how the process of the present invention is carried out, but it is to be understood that these examples are given by Way of illustration and not of limitation.

Example I 3425 g. of pyrogenic streptomycin hydrochloride, containing 787 million units of streptomycin,

was dissolved in 11.5 liters of pyrogen-free water and to this (approximately 23% of solution) was added 1700 g. (approximately 50% based upon weight of streptomycin hydrochloride) of powdered charcoal, preheated for eight hours at 250 C. The mixture was agitated for one hour and then filtered through a 10-inch Biichner funnel into a five gallon pyrex bottle. (All glassware was made pyrogen-free by heating for one hour at 250 C.) The charcoal was washed with two 1.5 liter portions of pyrogen-free water, which were combined with the main filtrate. The charcoal was then washed with about three to four gallons of water and this wash was worked up separately.

The combined filtrates were then sterilizedby filtration through a Seitz pad S-l, frozen, and dried from the frozen state under vacuum. The resulting dry streptomycin hydrochloride passed the U. S. P. pyrogen test at 10,000 u./kg.

The aqueous streptomycin hydrochloride recovered as washes can be used to d.ssolve another batch for pyrogen treatment.

Generally, the direct recovery of streptomycin hydrochloride is about 85% and the water washes The latter of course is recovered by recycling or by working up separate- 1y.

Example II Theree batches of streptomycin hydrochloride of different concentration were treated with charcoal, preheated for eight hours at 250 C., in the amounts shown in the table below, by mechanically stirring the charcoal suspension for one hour. The suspensions were then each filtered through a Biichner funnel using coarse filter paper over which a cloth was placed to facilitate removal'of the charcoal for re-slurrying. (All glassand ceramic-ware was made pyrogen-free by heating to 250 C. for one hour.) After each filtration, the charcoal was returned from the funnel to the original container and slurried with pyrogen-free water, equal in volume to 10% of the volume of the original solution, and again filtered. The charcoal for each batch was reslurried twice more as above described, and the original and three re-slurry filtrates from each batch were combined, and the charcoal set aside for recovery of residual adsorbed treptomycin.

The combined filtrates from each batch were sterilized by Seitz filtration, frozen, and dried from the frozen state under vacuum, yielding streptomycin hydrochloride which passed the U. S. P. pyrogen test at 10,000 u./kg.

Aliquots of each batch before charcoal tr tment and after charcoal treatment were dried, weighed, and assayed, yielding data which is included in the following table. (Concerning the activity changes, it is to be noted that it is difficult to evaluate changes of 10% or less by microbial assay except statistically on a large number of batches.)

Table Batch Item Cone. of solution treated 20% Weight streptomycin hydro- 72.0 g. 1,230 g 1,375 g.

chloride before charcoal treatment. Charcoal used (weight) 10.8 g 246 g 344 g. Charcoal used (per cent based on dry streptomycin hydrochloride). Weight of pyl'ogen-free strcp- 06.2 g. 1,105 g 1,275 g.

tomycin hydrochloride recovered directly. Weight loss 58g.=8% 1257g 100 g.=7%

l0 Activity before charcoal trent- 407 327........ an

ment (units/mg). Activity ofpyrogen-frce prod- 420 354 354 not (units/mg). Pyrogenicity Passes Passes Passes 1 U. S. P. standard test for pyrogens.

While the foregoing examples show the removal of pyrogen from streptomycin hydrochloride, it will be understood that pyrogens are removed from other streptomycin acid salts in the same manner by following the procedure described therein.

Modifications may be made in carrying out the present invention without departing from the spirit and scope thereof, and the invention is to be limited only by the appended claims.

We claim:

1. The process that comprises adding powdered charcoal to an aqueous solution of a pyrogenic streptomycin acid salt, agitating the mixture for about one hour, and filtering off the charcoal, thereby recovering a solution of the streptomycin acid salt free of pyrogens.

2. The process that comprises adding powdered charcoal, preheated for about eight hours at 250 C., to an aqueous solution of a pyrogemc streptomycin acid salt, agitating the mixture for about one hour, and filtering off the charcoal, thereby recovering a solution of the streptomycin acid salt free of pyrogens.

3. The process that comprises adding to an aqueous solution of a pyrogenic streptomycin acid salt, having a concentration of about 2 to 25%, an amount of powdered charcoal equal to 10 to of the weight of streptomycin acid salt in said solution, the higher percentage of charcoal being used with the higher concentration of said solution, agitating the mixture for about one hour, and filtering off the charcoal, thereby recovering a solution of the streptomycin acid salt free of pyrogens.

4. The process that comprises adding to an aqueous solution of a pyrogenfc streptomycin acid salt, having a concentration of about 2 to 25%, an amount of powdered charcoal, based upon the weight of streptomycin acid salt in said solution, varying from about 10 to 12% with a 2% solution to about 25 to 50% with a 25% solution, agitating the mixture for about one hour, and filtering off the charcoal, thereby recovermg a solution of the streptomycin acid salt free 9 P g ns.

5. The process that comprises adding powdered charcoal to an aqueous solution of a pyrogenic streptomycin acid salt, agitating the mixture for about one hour, filtering 011 the charcoal, reslurryinz and filtering the charcoal oneto three times with volumes of pyrogen-freewater equal to about 10% of the volume of the original solution, freezing the combined filtrates and drying from the frozen state under vacuum, thereby recovering the streptomycin acid salt free of pyrogens.

EUGENE E. HOWE. MAX TISHLER.

REFERENCES CIT The following references are of record in the file of this patent: a

6 UNITED STATES m'mu'rs Name Date Peck July 27, 1948 OTHER REFERENCES Carter et al.: Jour. Biol. Chem, vol. 160, pp. 337-342, Sept. 1945.

Le Page et al.: Jour. Biol. Chem, vol 162, pp. 163-171, 1946 (9 pages).

Mueller: JACS, vol. 69, (1947), pp. 195-200 (6 paces).

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1. THE PROCESS THAT COMPRISES ADDING POWDERED CHARCOAL TO AN AQUEOUS SOLUTION OF APYROGENIC STREPTOMYCIN ACID SALT, AGITATING THE MIXTURE FOR ABOUT ONE HOUR, AND FILTERING OFF THE CHARCOAL, THEREBY RECOVERING A SOLUTION OF THE STREPTOMYCIN ACID SALT FREE OF PYROGENS. 